The major functional domains of the glucocorticoid receptor (GR) include a sequence-specific DNA binding domain near the center of the polypeptide chain and a hormone binding domain in the Cterminal region. The aim of this proposal is to establish a detailed genetic map of the 250-amino acid hormone binding domain, by obtaining single amino acid substitutions that affect hormone binding affinity and/or specificity. This map will be essential for a complete understanding of the structure and function of the hormone binding domain, which determines the ligand affinity and specificity of the GR and regulates the activity of the GR protein as a conditional gene-specific transcriptional activator. The analysis will be performed using two complementary genetic approaches. First, a somatic cell genetics approach will make use of the glucocorticoid induced cytolytic response of the mouse lymphoma cell fine W7MG1 to select hormone-resistant cell lines containing mutant forms of the GR with altered hormone binding affinity and/or specificity. GR cDNA clones will be obtained from the mutant cell lines after polymerase chain reaction (PCR) amplification of single-stranded cDNA. Site directed mutagenesis will be performed in subregions dictated by the somatic cell genetics data and by biochemical data indicating that specific residues are involved in hormone binding. The mutations will be introduced by using PCR primed by synthetic oligonucleotides containing specifically designed point mutations. The phenotype of each mutant GR cDNA will be tested by co-transfecting the GR-negative COS-7 cell line with a GR-cDNA expression vector and a hormone responsive reporter gene, consisting of the chloramphenicol acetyl transferase (CAT) gene drive by the mouse mammary tumor virus promoter. The health relatedness of this project lies in the fact that steroid receptors play a pivotal role in steroid hormone action. In addition, a variety of malignant tumors treated clinically with steroid agonists and antagonists. The tumors frequently develop steroid resistance, which may arise from a subpopulation of the tumor cells that have sustained a mutation that alters hormone binding affinity and/or specificity. Understanding of this phenomenon will depend upon a detailed structure-function analysis of the hormone binding pockets of these steroid receptor proteins.